1. Technical Field
This invention relates to the application of genetic engineering techniques to plants. Specifically, the invention relates to compositions and methods for transformation of nucleic acid sequence into plant cell plastids.
2. Background
Molecular biological techniques have enabled researchers to introduce pieces of DNA from one organism to another organism. Such techniques, referred to as recombinant DNA technology, have positively impacted the areas of medicine and agriculture. Conventional cloning methods have enabled the introduction of new pharmaceuticals and improved crops of agricultural importance. As the need for the introduction of multiple pieces of DNA and larger fragments of DNA into numerous target hosts increases, the need for novel cloning strategies increases accordingly.
The plastids of higher plants are an attractive target for genetic engineering. Plant plastids (chloroplasts, amyloplasts, elaioplasts, chromoplasts, etc.) are the major biosynthetic centers that in addition to photosynthesis are responsible for production of industrially important compounds such as amino acids, complex carbohydrates, fatty acids, and pigments. Plastids are derived from a common precursor known as a proplastid and thus the plastids present in a given plant species all have the same genetic content. Plant cells contain 500-10,000 copies of a small 120-160 kilobase circular genome, each molecule of which has a large (approximately 25 kb) inverted repeat. Thus, it is possible to engineer plant cells to contain up to 20,000 copies of a particular gene of interest which potentially can result in very high levels of foreign gene expression.
Previous studies directed to stable transformation of plant chloroplasts have relied on homologous recombination to incorporate desired gene constructs into plastids using spectinomycin as the selectable marker for selection of transplastomic plants. However, at present, methods for multiple rounds of plastid transformation (for example for gene stacking) are restricted due to the limited number of selectable markers described for plastid transformation. Thus, there is a need in the art for methods employing additional selectable markers for obtaining plastid transformed plants.
By this invention, methods for obtaining plants having transformed plastids are provided. Specifically, methods are provided for obtaining plants having transformed plastids by selection of plastid transformed cells on medium containing a plastid lethal compound.
A first aspect of the present invention provides constructs useful for obtaining transplastomic plants. The constructs generally comprise a promoter functional in a plant cell plastid and a nucleic acid sequence encoding a protein that provides resistance to plastid lethal compounds.
Another aspect of the present invention is to provide methods for obtaining transplastomic plants using a nucleic acid sequence encoding a protein that provides resistance to plastid lethal compounds.
A further aspect of the present invention provides methods for obtaining transplastomic plants on selective medium containing glyphosate.
Also provided in the present application are methods for obtaining plastid transformed plants on selective medium containing phosphinothricin.
In yet another aspect of the present invention, methods for regenerating a plant having transformed plastids on medium containing plastid lethal compounds are provided.
The present invention also provides methods for the regeneration of a plant from a plant cell by growing the plant on medium containing plastid lethal compounds.
Another aspect of the present invention provides a method of obtaining a plastid transformed plant that does not contain an introduced nucleic acid sequence encoding a gene providing resistance to an antibiotic.
In a further aspect, methods for transforming the plastids of a host plant cell plastid are provided. The method generally comprises introducing into a plant cell plastid a first construct having a promoter functional in a plant cell plastid, a nucleic acid sequence encoding a protein that provides tolerance to a plastid lethal compound and a transcriptional termination region functional in a plant cell plastid. Additional expression cassettes can also be introduced, such cassettes having a promoter functional in a plant cell plastid, a nucleic acid sequence encoding a protein that provides tolerance to plastid non-lethal compounds, and a transcriptional termination region. Plant cells having the introduced construct or constructs are then grown on a first medium containing a plastid non-lethal compound for a time period sufficient to permit plastids containing the construct that provides resistance to a plastid lethal compound to increase in number, and then removed from the first medium and placed on a second medium containing a plastid lethal compound to permit selection of those transplastomic plant cells expressing the protein conferring tolerance to the plastid lethal compound.
In yet a further aspect, methods for transforming the plastids of a host plant cell plastid are provided. The method generally comprises introducing into a plant cell plastid a first construct having a promoter functional in a plant cell plastid, a nucleic acid sequence encoding a protein that provides tolerance to a plastid lethal compound and a transcriptional termination region functional in a plant cell plastid. Plant cells having the introduced construct are then grown on a first medium containing a sublethal concentration of a plastid lethal compound for a period of time sufficient to permit transformed plastids in transplastomic plant cells to increase in number as compared to non-transformed plastids, and then removed from such first medium and placed on a second medium containing a lethal concentration of a plastid lethal compound to permit selection of those transplastomic plant cells expressing the protein conferring tolerance to the plastid lethal compound.
Also considered in the present invention are the plant cells and plants produced by the methods described herein.